Alexa fluor 555 goat antirat secondary antibody

The method previously described was used (58 ). Briefly, female C57BL6 mice (8–12 weeks old) were i.v. injected with 2.7 μg of spike in 50 μl of PBS. After 4 h, animals were euthanized by cervical dislocation, intracardially perfused with 10 ml of PBS, to wash nonadhered leukocytes, followed by perfusion with 5 ml fluorescent isothiocyanate–labeled concanavalin A (ConA) lectin (40 μg ml⁻¹ in PBS, pH 7.4; Invitrogen), to label adherent leukocytes and EC. Residual unbound ConA was then removed by perfusion with 10 ml of PBS. Eyes were fixed in 4% PFA for 10 min, retinas dissected, maintained in cold methanol for at least 20 min, washed, permeabilized, and blocked with PBS containing 1% Triton X-100, 0.4% BSA, and 10% normal goat serum for 48 h at 4 °C. Retinas were then immunostained for 48 h at 4 °C with a 1:200 dilution of anti-CD45 mAb (Santa Cruz), washed, and labeled overnight with a 1:500 dilution of Alexa Fluor 555 goat antirat secondary antibody (Invitrogen). Finally, retinas were washed, flat mounted, and cover slipped using Vectashield H-1000 (Vector Laboratories, Inc). Retinal vessels and intravascular ConA+ and CD45+ cells, identified as leukocytes, were digitized under a fluorescence microscope.

Mice were sacrificed at the indicated time points, and corneas were excised, rinsed in PBS, and incubated in 20 mM EDTA in PBS for 30 min at 37 °C for removal of the corneal epithelium. Subsequently, corneas were washed, fixed in ethanol, and blocked in 2% BSA. For the analysis of corneal macrophages, BV, and LV, corneas were stained with FITC-conjugated anti-CD31 (BD Biosciences, Heidelberg, Germany) and anti-LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1) (Acris Antibodies GmbH, Herford, Germany), or anti-F4/80 (Invitrogen, Eugene, USA) overnight. To detect LYVE-1 or F4/80 corneas were washed and incubated with Cy3 goat-anti rabbit (Dianova, Hamburg, Germany) or Alexa fluor 555 goat anti-rat secondary antibody (Invitrogen, Eugene, USA) for 45 min. After final washing, corneas were mounted on microscope slides with fluorescence mounting medium (Dako, Glostrup, Denmark).